CTAB for a chloroplast enriched extraction - differential centrifugation for small quantities of material? (Oct/01/2009 )

Hi, I am doing an old-fashioned CTAB extraction from small quantities of plant material and need to enrich the extraction for chloroplast DNA. I'm not trying to get pure chloroplast DNA, just a significantly enriched extract. I've been advised to do a simple differential centrifugation step after cell disruption (I'm grinding the samples in eppendorf tubes in liquid N). The recipes I have seen for differential centrifugation involve suspension in a buffer (50mM Tris-HCL ph 8.0, 10mM EDTA, 20% sucrose, 5mM 2-mercaptoethanol, 0.1% BSA) followed by passing through cheese cloth, prior to the differential centrifugation. Problem is I don't have cheesecloth and in any case, I think the samples are too small for this.

What I have tried so far is grinding the samples in liquid N, vortexing the powder in the above buffer in an eppendorf tube, and then going straight onto centrifugation at 1000g for few seconds. I then remove as much supernatant as possible and repeat the 1000g centrifugation step (the first step is just to remove the large fragments). The supernatant from this then gets centrifuged at 3000g for 10mins to precipitate the chloroplasts as a pellet. I remove the supernatant and then go straight to a standard CTAB extraction with this pellet.

Problem is I'm getting very little DNA, I'm guessing because most of the chloroplasts aren't in suspension after the 10g centrifugation but rather still trapped down in the cellular gunge.. Also, I'm worried about potential degradation of the DNA during this process... or can I assume that prior to using CTAB the chloroplast membranes are still intact and so the DNA I want is protected from nucleases etc? If so, would it help if I incubated the suspension in the buffer for a while before differential centrifugation?

Given the small quantities I am dealing with, could I use something else instead of cheese cloth, e.g. a filter from an extraction kit? I don't want to use anything that is going to degrade the quality of the DNA.

Thanks.

-smoo-

try doing s sucrose or other gradient centrifugation to separate out the gunk from the sub-cellular components.

-bob1-

Hi, I developed a method for obtaining cpDNA for chlorophyllus cells from B. gracilis with CTAB, to increase the amount of cpDNA I first added more green material, alo treated with CTAB/NaCl after disrupting the organelles, if you need help send me a message.